Glycodelin A, not glycodelin S, is apoptotically active. Relevance of sialic acid modification.

Debaditya Mukhopadhyay,Swathi Sundarraj,Anjali A Karande,Anshula Alok

doi:10.1074/jbc.m306673200

Debaditya Mukhopadhyay, Swathi Sundarraj + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m306673200

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Abstract

Glycodelin, previously known as PP14 (placental protein-14), is a kernel lipocalin secreted by the glandular epithelium of the endometrium upon progesterone stimulation and by the seminal vesicles. The isoform of the protein present in female reproductive tissue, glycodelin A (GdA), and the male counterpart, glycodelin S (GdS), have identical amino acid sequences, but strikingly different N-linked glycans. It is well documented in literature that GdA is an immunosuppressive protein, and we have shown that this activity is due to its ability to induce apoptosis in activated T cells. The precise role of GdS in seminal plasma is not known. In this study, we report that GdS is not apoptotically active. We observe that the apoptotic activity requires the presence of sialic acid residues on the complex glycans, as in the case of GdA; however, complex glycans of GdS are non-sialylated. We have expressed the wild-type protein in Pichia pastoris, which does not add sialic acid to the secreted proteins, and confirmed our observations that the protein is apoptotically inactive in the non-sialylated form. Our results indicate that differential glycosylation modulates the function of the different glycodelin isoforms.

Highlights

Glycodelin, known as PP14 [1], is a 162-amino acid glycoprotein classified under the lipocalin superfamily based on its 70% similarity to equine ␤-lactoglobulin [2]
1 The abbreviations used are: GdA, glycodelin A; GdS, glycodelin S; Peripheral blood mononuclear cells (PBMCs), peripheral blood mononuclear cell; mAb, monoclonal antibody; PBS, phosphate-buffered saline; Pic-fl-Gd, full-length glycodelin expressed in Pichia pastoris; Ni2ϩ-NTA, nickel-nitrilotriacetic acid; PNGase F, peptide N-glycosidase F; Endoglycosidase H (Endo-H), endoglycosidase H; FACS, fluorescence-activated cell scan; t-pic-Gd, N-terminal 23-amino aciddeleted glycodelin expressed in P. pastoris
GdS purified by the identical procedure did not show inhibition of mAb OKT3-induced PBMC proliferation (Fig. 2B, panel a) or Jurkat cell proliferation

Summary

The abbreviations used are

GdA, glycodelin A; GdS, glycodelin S; PBMC, peripheral blood mononuclear cell; mAb, monoclonal antibody; PBS, phosphate-buffered saline; Pic-fl-Gd, full-length glycodelin expressed in Pichia pastoris; Ni2ϩ-NTA, nickel-nitrilotriacetic acid; PNGase F, peptide N-glycosidase F; Endo-H, endoglycosidase H; FACS, fluorescence-activated cell scan; t-pic-Gd, N-terminal 23-amino aciddeleted glycodelin expressed in P. pastoris. The steroid dependence, together with its immunosuppressive activity, suggests that GdA plays a role in down-modulating the T cell-mediated immune response against the fetal allograft in the maternal system. Complete digestion of GdA with trypsin indicated that the isolated glycopeptides are not active and that the protein backbone is necessary for inducing apoptosis. Both glycodelins were deglycosylated using various glycosidases, and loss of GdA’s apoptotic activity was observed when the glycoprotein was desialylated. Both the sialic acid residues on the N-linked glycans and the protein backbone are required for native GdA to show apoptotic activity

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION